Compounds and their salts specific to the PPAR receptors and the EGF receptors and their use in the medical field

ABSTRACT

Therefore the present invention relates specifically to the compounds of general formula (I), in which R 1  and R 2 , which may be identical or different, are selected from the group comprising H, —C n H 2n-1 , a linear or branched alkyl group having from 1 to 6 carbon atoms, or together form an aromatic or aliphatic ring with 5 or 6 atoms; R 3  is selected from —CO—CH 3 , —NHOH, —OH, —OR 6  in which R 6  is a linear or branched alkyl group having from 1 to 6 carbon atoms; R 4  is selected from H, a linear or branched alkyl group having from 1 to 6 carbon atoms, phenyl, benzyl, —CF 3  or —CF 2 CF 3 , vinyl or allyl; R 5 , R 7 , R 8  are hydrogen atoms; or R 3  and R 4 , R 4  and R 5 , or R 7  and R 8  together form a ring, fused to the benzene, aromatic or aliphatic ring with 5 or 6 atoms comprising from 1 to 2 heteroatoms selected independently from the group comprising N, O. and use thereof in the medical field.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/IE2006/000076, filed 24 Jul. 2006, published in English, andclaims priority under 35 U.S.C. §119 or 365 to Italian Application No.RM2005A000390, filed 22 Jul. 2005.

FIELD OF THE INVENTION

The present invention relates to compounds and their salts specific tothe PPAR receptors and the EGF receptors and their use in the medicalfield.

OBJECT OF THE INVENTION

The compounds and their salts according to the present invention can beused advantageously for the prevention and treatment of tumoursexpressing the PPARγ receptors (Peroxisome Proliferator-ActivatedReceptors) and the EGF receptors (Epidermal Growth Factor receptors)such as tumours of the: oesophagus, stomach, pancreas, colon, prostate,breast, uterus and appendages, kidneys and lungs. Moreover, thecompounds and their salts according to the invention can be used for thetreatment of chronic inflammatory diseases, in particular chronicintestinal diseases, such as Crohn's disease and ulcerativerectocolitis.

BACKGROUND TO THE INVENTION

The PPARγ receptors are nuclear receptors (group of approx. 50transcription factors) which control the expression of many genes thatare important for the regulation of lipid metabolism, the synthesis ofinsulin and the processes of carcinogenesis and inflammation (Bull A W,Arch Pathol Lab Med 2003; 127: 1121-1123) (Koeffler H P, Clin Cancer Res2003; 9: 1-9) (Youssef J et al., J Biomed Biotec 2004; 3: 156-166).

There are various natural and synthetic agonists which bind to the PPARγreceptors and alter their conformation, giving rise to activation.Natural and synthetic ligands are described in The Lancet 2002;360:1410-1418.

Recent studies have shown that treatment of tumour cells with ligands ofthe PPARγ receptors induces a decrease in cellular proliferation, celldifferentiation and apoptosis, suggesting potential application of suchcompounds as agents for preventing carcinogenesis (Osawa E et al.,Gastroenterology 2003; 124:361-367).

Other studies have shown that ligands of the PPARγ receptors (e.g.troglitazone) have anti-inflammatory effects and inhibit the mucosalinflammatory response in animal models of IBD (Tanaka T et al., CancerRes 2001; 61: 2424-2428).

Moreover, evidence has been published very recently that the intestinalanti-inflammatory activity of 5-ASA (5-aminosalicylic acid, mesalazine),the gold standard in the treatment of IBD, is dependent on binding, andconsequent activation, of the PPARγ receptors (Rousseaux C et al., J ExpMed 2005; 201: 1205-1215).

The transmembrane receptor with tyrosine-kinase EGF activity isexpressed to a very high degree in activated form in various types ofneoplasms (Mendelsohn J, Endocr Relat Cancer 2001; 8: 3-9) (Harari P M,Endocr Relat Cancer 2004; 11: 689-708).

Overexpression of the receptor is also related to potential ability ofcarcinomatous cells to metastasize. In connection to this, it has beendemonstrated that EGF promotes the migration and invasiveness of variouscell types connected with lesions at the level of interactions with theextracellular matrix (Brunton et al., Oncogene 1997; 14: 283-293).

Numerous studies performed both on experimental animals and in man haveestablished the efficacy of inhibitors of the EGF receptor incontrolling proliferation and the spread of tumours (Mendelsohn J,Endocr Relat Cancer 2001; 8: 3-9) (Harari P M, Endocr Relat Cancer 2004;11: 689-708).

There is no doubt that the intracellular signals triggered by activationof the EGF receptor facilitate the growth and survival of neoplasticcells, contributing to the development of the pathology, and that suchsignals are essential in determining the ability of tumour cells tospread and colonize remote organs (Mendelsohn J, Endocr Relat Cancer2001; 8: 3-9) (Kari C et al., Cancer Res 2003; 63: 1-5).

From the foregoing and bearing in mind, moreover, that from thebiological standpoint, chronic inflammatory processes play a part incarcinogenesis, it becomes clear that there is a real need forinnovative research into new chemical entities which, by theircomplementary action both on the PPARγ receptors and on the EGFreceptors, are able to exert anti-inflammatory and anti-tumour action,of the chemo-preventive, anti-proliferative and anti-metastatic type.

SUMMARY OF THE INVENTION

The present invention relates to novel and inventive medical andtherapeutic uses of a series of compounds In so far as any of thesecompounds are not known, the invention also relates to these compounds.

The present invention provides a novel class of compounds that aresuitable for the prevention and treatment of cancer and of chronicinflammation by the modulation of specific receptors such as the PPARγreceptors and the EGF receptors.

Therefore the present invention relates specifically to the compounds ofgeneral formula (I)

in whichR₁ and R₂, which may be identical or different, are selected from thegroup comprising H, —C_(n)H_(2n-1), a linear or branched alkyl grouphaving from 1 to 6 carbon atoms, or together form an aromatic oraliphatic ring with 5 or 6 atoms;R₃ is selected from —CO—CH, —NHOH, —OH, —OR₆ in which R₆ is a linear orbranched alkyl group having from 1 to 6 carbon atoms;R₄ is selected from H, a linear or branched alkyl group having from 1 to6 carbon atoms, phenyl, benzyl, —CF₃ or —CF₂CF₃, vinyl or allyl; R₅, R₇,R₈ are hydrogen atoms;orR₃ and R₄, R₄ and R₅, or R₇ and R₈ together form a ring, fused to thebenzene, aromatic or aliphatic ring with 5 or 6 atoms comprising from 1to 2 heteroatoms selected independently from the group comprising N, O.

The invention also relates to the specific subgroup of compounds ofgeneral formula (Ia)

in whichR₁ and R₂, which may be identical or different, are selected from thegroup comprising H, —CO—CH₃, —C_(n)H_(2n-1), a linear or branched alkylgroup having from 1 to 6 carbon atoms, or together form an aromatic oraliphatic ring with 5 or 6 atoms;R₃ is selected from —NHOH, —OH, —OR₆ in which R₆ is a linear or branchedalkyl group having from 1 to 6 carbon atoms;R₄ is selected from —H, a linear or branched alkyl group having from 1to 6 carbon atoms;R₅, R₇, R₅ are hydrogen atoms;orR₃ and R₄, R₄ and R₅, or R₇ and R₈ together form a ring, fused to thebenzene, aromatic or aliphatic ring with 5 or 6 atoms comprising from 1to 2 heteroatoms selected independently from the group comprising N, O.

The aforementioned linear or branched alkyl group of formula (I) or(IIa) having from 1 to 6 carbon atoms can be selected from —CH₃, —C₂H₅,isopropyl, propyl, C_(n)H_(2n-1).

The present invention also relates to compounds as recited in formulae(I) and (Ia) except without allowing for where R₃=—COCH₃. There is atheoretical possibility that some acetyl derivatives such as at leastsome of those when R₃=—COCH₃ may be inactive, since N-acetylation is themetabolic detoxification system for aromatic amines. The theoreticalpossibility arises from the observation that the inactive metabolite of5-ASA is N-acetyl 5-ASA.

In some embodiments of both formula (I) and (Ia) the invention, R₇ andR₈ may not form a ring. Thus R₃ and R₄ or R₄ and R₅ may together form aring, fused to the benzene, aromatic or aliphatic ring with 5 or 6 atomscomprising from 1 to 2 heteroatoms selected independently from the groupcomprising N, O. In some embodiments compounds 29, 36 and 37 areexcluded. In some embodiments of both formula (I) and (Ia) theinvention, R₇ and R₈ may not form a ring except when R₄ is CH₃. In someembodiments of both formula (I) and (Ia) the invention, R₇ and R₈ maynot form a ring when R₄ is selected from H. In some embodiments, theinvention relates to the ketolenes provided by the invention. In someembodiments, compound 29 is excluded.

In some embodiments, one or more compounds selected from the groupconsisting of compounds 5, 6, 7, 8, 9, 12, 16, 18, 19, 24, 25, 27, 30,and 41 are excluded.

In some embodiments of both formula (I) and (Ia) the invention, R₁ andR₂ may not form a ring. Thus R₁ and R₂, which may be identical ordifferent, may be selected from the group comprising —H, —C_(n)H_(2n-1),a linear or branched alkyl group having from 1 to 6 carbon atoms. Insome embodiments compound 41 is excluded.

In some embodiments of both formula (I) and (Ia) of the invention, R₄may not be branched. Thus R₄ may be selected from H, a linear alkylgroup having from 1 to 6 carbon atoms; R₅, R₇, R₈ are hydrogen atoms. Insuch embodiments, compound 30 and 31 are excluded. In some embodiments,R₄ may not be branched when the amino group is at position 4′ on thephenyl ring. In such embodiments, compound 30 is excluded. In someembodiments, the linear alkyl group may have only 1 carbon atom (i.e.,CH₃).

In some embodiments of both formula (I) and (Ia) the invention, R₁ andR₂ are —H. In some embodiments compounds 6, 9, 24, 27, 38 and 41 areexcluded.

In some embodiments of both formula (I) and (Ia) of the invention, R₂may not be different to R₁.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —OH, R₄ is selected from the group consisting —H, a branched alkylgroup having from 1 to 6 carbon atoms, or R₃ and R₄, together form aring, fused to the benzene, aromatic or ring with 5 or 6 atomscomprising from 1 to 2 heteroatoms selected independently from the groupcomprising N, O. In some embodiments, the branched alkyl group may be—CH(CH₃)₂. In some embodiments, the branched alkyl group may be—CH(CH₃)₂ at the R₈ position. In some embodiments, R₃ and R₄ form a5-membered aliphatic ring with a single O atom. In some embodiments(such as the ones described in this paragraph), compounds 7, 8, 18, 19,42 are excluded.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —OH and R₄ is —H, the group NR₁R₂ cannot be at the 4′ position(and should be at R₅ or R₈). In some embodiments, this may beparticularly the case where R₁ and R₂ are —CH₃. In some embodimentscompound 24 is excluded.

In some embodiments of both formula (I) and (Ia) of the invention, R₁and R₂ are the same. In some embodiments compounds 9 and 12 areexcluded.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —OH and R₄ is —H, the group —NR₁R₂ cannot be at the R₅ (and shouldbe at the R₈ position). In some embodiments, this may be where R₁ and R₂are —H. In some embodiments compound 6 is excluded.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —NHOH, R₄ is a linear or branched alkyl group having from 1 to 6carbon atoms, (or, if formula (I) phenyl, benzyl, —CF₃ or —CF₂CF₃, vinylor allyl), R₃ and R₄, together form a ring, fused to the benzene,aromatic or aliphatic ring with 5 or 6 atoms comprising from 1 to 2heteroatoms selected independently from the group comprising N, O. Insome embodiments compound 7 is excluded.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —NHOH, and R₄ is a linear or branched alkyl group having from 2carbon atoms, the group —NR₁R₂ may not be at the 4′ position and can beat the R₈ position. In some embodiments compound 25 is excluded.

In some embodiments of both formula (I) and (Ia) of the invention, whenR₃ is —NHOH, and R₄ is —H, the group —NR₁R₂ may not be at R₈ and must beat the 4′ position. In some embodiments compound 5 is excluded.

According to one embodiment, R₃ and R₄ of the compounds of formula (I)and (Ia) can form a ring according to the following formula (II)

while R₁, R₂, R₅, R₇ and R₈ are defined above.

According to another embodiment R₄ and R₅ of the compounds of formula(I) and (Ia) can form a ring according to the following formula (III)

while R₁, R₂, R₃, R₇ and R₈ are defined above.

According to a further embodiment R₇ and R₈ of the compounds of formula(I) and (Ia) can form a ring according to the following formula (IV) or(V)

while R₁, R₂, R₃, R₄ and R₅ are defined above.

In particular, the compounds of formula (I) and (Ia) according to thepresent invention can be selected from the group comprising

-   4-amino-N-hydroxy-2-methoxybenzamide (compound 13)-   5-amino-N-hydroxy-2-methoxybenzamide (compound 14)-   5-amino-2,3-dihydrobenzofuran-7-carboxylic acid (compound 17)-   5-amino-2-ethoxy-N-hydroxybenzamide (compound 26)-   6-amino-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one (compound 28)-   1,2,3,4-tetrahydro-6-hydroxyquinoline-5-carboxylic acid (compound    29)-   5-amino-2-isopropoxybenzoic acid (compound 31)-   6-methoxy quinoline-5-carboxylic acid (compound 36)-   6-methoxy-1,2,3,4-tetrahydroquinoline-5-carboxylic acid (compound    37)-   5-diisopropylaminosalicylic acid (compound 38)-   4-diisopropylaminosalicylic acid (compound 42).

The present invention also provides for compounds wherein R₁ and R₂, areselected from the group consisting of —H and —CH(CH₃)₂. R₁ and R₂ mayboth be identical. In some embodiments, R₁ and R₂ may be —CH(CH₃)₂.

One example comprises the following structure (compound 38):

In other embodiments of the invention, R₁ and R₂, are both —H.

The present invention also provides for compounds wherein R₃ is selectedfrom the group consisting of —NHOH and —OH. In some embodiments R₃ maybe —NHOH. One example comprises the following structure (compound 13):

A further example comprises the following structure (compound 14):

A further example comprises the following structure (compound 26):

In some embodiments of the invention, R₃ may be —OH.

A suitable example comprises the following structure (compound 17):

A further example comprises the following structure (compound 31):

In some embodiments of the invention, R₄ may be —H. In some embodimentsof the invention, R₄ may be CH₃. In some embodiments of the invention,R₄ may be, —CH₂CH₃. In some embodiments of the invention, R₄ may be—CH(CH₃)₂.

A further example comprises the following structure (compound 28):

In some embodiments of the invention, R₃ and R₄ may together form analiphatic ring, fused to the benzene, of 5 or 6 atoms comprising onehetero atom O (oxygen).

The compounds according to the invention can be used advantageously inthe medical field.

Therefore the present invention further relates to a pharmaceuticalcomposition comprising one or more compounds according to the inventionas active principles in combination with one or more pharmaceuticallyacceptable excipients or adjuvants.

Furthermore, the present invention relates to the use of the compoundsaccording to the invention for the preparation of a medicinal productfor the prevention and treatment of tumours expressing PPARγ receptorsand EGF receptors such as tumour of the oesophagus, stomach, pancreas,colon, prostate, breast, of the uterus and appendages, of the kidneysand of the lungs.

Moreover, the invention relates to the use of the compounds according tothe present invention for the preparation of a medicinal product for thetreatment of chronic inflammatory diseases such as Crohn's disease andulcerative rectocolitis. The present invention also relates to methodsof treatment of humans and/or mammals (including rodents, farm animals,domestic pets, mice, rats, hamsters, rabbits, dogs, cats, pigs, sheep,cows, horses).

In particular, apart from the specific compounds mentioned above, thefollowing compounds can be used for the applications described above:

-   5-aminosalicylo-hydroxamic acid (compound 5)-   3-dimethylaminosalicylic acid (compound 6)-   2-methoxy-4-aminobenzoic acid (compound 7)-   2-methoxy-5-aminobenzoic acid (compound 8)-   5-methylaminosalicylic acid (compound 9)-   4-methylaminosalicylic acid (compound 12)-   4-acetylaminosalicylic acid (compound 16)-   2-ethoxy-4-aminobenzoic acid (compound 18)-   2-ethoxy-5-aminobenzoic acid (compound 19)-   4-dimethylaminosalicylic acid (compound 24)-   2-ethoxy-4-aminobenzoylhydroxamic acid (compound 25)-   6-hydroxyquinoline-5-carboxylic acid (compound 27)-   2-(2-propyl)oxy-4-aminobenzoic acid (compound 30)-   4-(1-piperazinyl)salicylic acid (compound 41).

The molecules of the present invention were derived from molecularmodeling work using mesalazine as a basis and all chemically feasiblevariations were evaluated in order to achieve the best score (affinityand activation of the receptor) in computer docking experiments.Consequently, it is believed that the compounds of the present inventionthat show comparable function and/or activity to mesalazine do sothrough similar biological pathways. It is believed that similarcharacteristics to mesalazine inherent in the molecules of the inventionconfer upon the molecules a similar activity in relation to the EGFpathway.

The experiments given herein make good models for use in the predictionof the use of the compounds in the various medical fields alreadydiscussed. The models used give meaningful results regardless of themechanism of action.

In addition to the above mentioned compounds, the present inventionprovides for the use of the following compounds (compound number followsprefix “2_”):

The present invention will now be described for purposes ofillustration, but without limiting it, according to its preferredembodiments, with particular reference to the diagrams in the appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

FIG. 1A shows the structures of compounds 13, 14, 17, 26, 31 and 38.

FIG. 1B shows the activation of the PPARγ receptors by molecules 17 and31 relative to cells treated with 5-ASA and rosiglitazone. The HT-29 STDcells transfected with the response element for PPARγ (2XCYP) andtreated with molecules 17 and 31 (30 mM) show induction of about twicethe activity of the reporter gene indicating the capacity of 5-ASA andnovel compounds 17 and 31 to induce activation of the PPARγ; the resultsare expressed as factor of increase in activation (mean±SEM) relative tothe untreated cells. The activations for compounds 13, 14, 26 and 38 arealso shown.

FIG. 2 shows the increase in expression of the PPARγ protein by theepithelial cells induced by compounds 17, 26 and 31; the level ofexpression of the PPARγ protein was evaluated by western blot inuntreated HT-29 cells (control) and after 24 hours of treatment with thenovel compounds or 5-ASA (30 mM) or rosiglitazone (10⁻⁵M) used aspositive controls. The values of optical density of the PPARγ werecalculated for each condition in proportion to the quantity of theβ-actin internal control in the same sample.

FIG. 3 shows the inhibition of proliferation of epithelial cells bycompounds 17 and 31; similarly to 5-ASA (30 mM) and to rosiglitazone(10⁻⁵M), compounds 17 and 31 inhibit proliferation of the HT-29 STDcells, tested by staining with nuclear Ki-67 (light grey) relative tothe cells incubated with the culture medium only (control); the nucleiwere stained blue (dark grey in figure) with Hoechst 33342 solution; theresults are expressed as average number of cells counted in oneexperiment.

FIG. 4 shows the induction of apoptosis of epithelial cells by compound31; similarly to 5-ASA (30 mM) and rosiglitazone (10⁻⁵M), molecule 31induced apoptosis identified in the TUNEL assay in HT-29 STD cells.Spontaneous apoptosis of 3% was observed in the untreated HT-29 cells;the results are expressed as average number of cells counted in oneexperiment.

FIG. 5A shows docking simulation of the manner of binding of variouscompounds to the PPARγ; compared with the interaction of the novelcompounds (from 13 to 38) stained according to the type of atom in theligand binding domain (LBD) represented by a white surface in the X-raycrystal structure of the PPARγ; description of the key hydrogen bindinginteractions between the novel molecules and the PPARγ.

FIG. 5B shows a detailed docking simulation of the manner of binding ofmesalazine to the PPARγ receptor.

FIG. 5C shows a more detailed docking simulation of the manner ofbinding of compound 17 to the PPARγ receptor.

FIG. 5D shows a more detailed docking simulation of the manner ofbinding of compound 31 to the PPARγ receptor.

FIG. 6 shows the analysis of PPARγ activity of compounds 13, 14, 17, 26,28, 31 and 38, in transfected HT-29 cells. This shows that the newmolecules increase the reporter gene activity, thereby displaying anactivity similar or superior to 5-ASA.

FIGS. 7-8 show the effect of the specified substances on theproliferation of three different human colon carcinoma cell lines (i.e.HT29, HT115 and DLD1). The cells were treated with increasingconcentrations of substances (0.5-10 mM)) for 48 hours and theproliferation was determined by using a colorimetric assay for themeasurement of BrdU incorporation. The optical density (OD) wasdetermined at 450 nm using an ELISA reader. Data indicate the mean±SD of3 separate experiments.

FIG. 9 shows the flow cytometric analysis of compound 14's inhibitoryeffect on the proliferation on DLD-1 cells. One of three representativeexperiments in which similar results were obtained is shown. Cells werelabeled with CFSE and their proliferative fraction was calculated after48 hours culture by flow cytometry.

FIG. 10A shows that compound 14 significantly enhances DLD1 cell deathat concentrations of 1.5 (p<0.01) and 3 mM (p<0.001). DLD1 were eitherleft unstimulated (Unst) or treated with compound 14 for 48 hours. Dataexpress mean±SD of 3 separate experiments and indicate the percentage ofcell death as assessed by FACS analysis of AV and/or PI-positive cells.

FIG. 10B shows Z-VAD, a pan-caspase inhibitor, reverses the effect ofcompound 14 on DLD-1 cell death. The results are displayed asbiparametric histograms of Annexin-V-FITC and PI fluorescences allowingdiscrimination between viable cells, apoptotic cells with an intactmembrane and cells undergoing secondary necrosis. One of threerepresentative experiments in which similar results were obtained isshown.

Table 1. Percentages of DLD-1 cell inhibition by graded doses (0.5-10mM) of the specified compounds. Cells were cultured in the presence orabsence of the compounds, and cell growth was then assessed by thecalorimetric (BrdU) assay after 48 hours culture.

EXAMPLE 1 Method of Preparing 4-Amino-N-hydroxy-2-methoxybenzamide(Compound 13)

Methyl 2-methoxy-4-aminobenzoate (10 g, 55.25 mmol) and hydroxylaminehydrochloride (15.36 g, 221 mmol) were taken up in MeOH (80 ml) and asolution of KOH (15.4 g, 275 mmol) in MeOH (55 ml) was added carefully.The resultant mixture was stirred at reflux for 36 hrs. The volatileswere removed in vacuo. The residue was taken up in 1M NaOH (50 ml) andwashed with ethyl acetate (EtOAc, 50 ml). Concentrated HCl was addedslowly until precipitation of a solid (pH was 10). The solid wasfiltered off, washed with H₂O, then methyl-tert-butyl ether (MTBE) anddried under vacuum. ¹H NMR showed the solid contained approx. 1/3 molarequivalent of ethyl acetate. The solid was taken up in 1M NaOH (100 ml)and the EtOAc was removed in vacuo. Concentrated HCl was added carefullyuntil precipitation of a solid (pH was 8). The solid was collected byfiltration, washed with H₂O, then MTBE and dried under vacuum to give4.67 g (47%) of the title compound as a dark red solid.

¹H NMR (δ, 250 MHz, d₆-DMSO): 3.75 (s, 3-H, OMe), 5.64 (s, 2-H, NH₂),6.14 (dd, 1-H, aromatic), 6.18 (brs, 1-H, aromatic), 7.48 (d, 1-H,aromatic), 8.73 (s, 1-H, NH), 10.06 (s, 1-H, OH).

EXAMPLE 2 Method of Preparing 5-Amino-N-hydroxy-2-methoxybenzamide(Compound 14)

Methyl 2-methoxy-5-nitrobenzoate (10 g, 47.39 mmol) and hydroxylaminehydrochloride (13.17 g, 189.56 mmol) were taken up in MeOH (100 ml) anda solution of KOH (13.27 g, 236.95 mmol) in MeOH (55 ml) was addedcarefully. Note—an exothermic reaction was observed; solids initiallydissolved, then a solid precipitated out of solution. TLC (run in EtOAc)of an acid washed aliquot showed a trace of the starting benzoate and anew product. H₂O (100 ml) was added and any undissolved solid collectedby filtration off, washed with isopropyl alcohol (IPA) and dried invacuo to give 10.23 g (>100%) of N-hydroxy-2-methoxy-5-nitrobenzamide asa white solid.

¹H NMR (δ, 250 MHz, MeOD): 4.18 (3-H, OMe), 7.43 (d, 1-H, aromatic),8.39 (dd, 1-H, aromatic), 8.83 (d, 1-H, aromatic)

N-Hydroxy-2-methoxy-5-nitrobenzamide (approx. 47.39 mmol) was taken upin IMS (500 ml) and 10% Pd on C (50% wet) (1 g) was added. The mixturewas hydrogenated at 50 psi for 1.5 hr, then filtered through celite, andthe celite washed with 2M NaOH (200 ml). The MeOH was removed in vacuoand the resulting aqueous residue washed with MTBE (100 ml). The pH ofthe aqueous was lowered to 7 by the careful addition of concentratedHCl, and the resulting precipitated solid collected by filtration,washed with H₂O, then MTBE and dried under high vacuum overnight to give5.33 g of the title compound (62%) as a light brown solid.

¹H NMR (δ, 250 MHz, d₆-DMSO): 4.09 (3-H, OMe), 7.02 (1-H, aromatic),7.18 (1-H, aromatic), 7.28 (1-H, aromatic), 9.36 (1-H, NH), 10.83 (1-H,OH).

EXAMPLE 3 Method of Preparing5-Amino-2,3-dihydrobenzo[b]furan-7-carboxylic Acid (Compound 17)

Nitric acid (9 ml) was added to a cooled (ice bath) solution of2,3-dihydrobenzo[b]furan-7-carboxylic acid (5.1 g, 31 mmol) intrifluoroacetic acid (45 ml). After 4 hours the mixture was quenchedinto ice-water (150 ml) and the mixture filtered to give5-nitro-2,3-dihydrobenzo[b]furan-7-carboxylic acid, which was washedwith water and used crude (wet) in the next stage.

¹H NMR (δ, 250 MHz, CD₃OD): 3.70 (2H, t, 8.7 Hz), 5.21 (2H, t, 8.9 Hz),8.67 (1H, d, 2.7 Hz), 8.83 (1H, d, 2.4 Hz)

The crude 5-nitro-2,3-dihydrobenzo[b]furan-7-carboxylic acid wasdissolved in the minimum amount of methanol (1.3 L) and nitrogen wasbubbled through the solution for 20 minutes. A mixture of 5% palladiumon charcoal (1.0 g) in water (20 ml) was added and the mixture loadedinto a 2 L autoclave. After 2 sequential evacuation-N₂ purges the vesselwas charged to 5 bar H₂ and stirred for 4 days. The mixture was flushedwith nitrogen, filtered through celite and concentrated to give5-amino-2,3-dihydrobenzo[b]furan-7-carboxylic acid (3.5 g, 19.5 mmol,63% over 2 steps)

¹H NMR (δ, 250 MHz, CD₃OD): 3.46 (2H, t, 8.5 Hz), 4.85 (2H, t, 8.7 Hz),7.19 (1H, br s), 7.27 (1H, br s)

EXAMPLE 4 Method of Preparing 5-Amino-2-ethoxy-N-hydroxybenzamide(Compound 26)

Step 1

Methyl 2-hydroxy-5-nitrobenzoate (11.82 g, 60 mmol), triphenylphosphine(17.29 g, 66 mmol) and EtOH (3.03 g, 3.85 ml, 66 mmol) were taken up inTHF (150 ml) and cooled in ice. Di-isopropyl-azodicarboxylate (8.31 g,10 ml, 66 mmol) was added carefully, and the reaction was stirred at RTfor 1 hr. The reaction was concentrated in vacuo, and the residue wastriturated with EtOAc (50 ml). The solid thus formed was collected byfiltration, washed with MTBE and dried in vacuo to give 12.2 g of crudemethyl 2-ethoxy-5-nitrobenzoate. The filtrate was evaporated in vacuo,and the resulting residue was triturated with EtOAc (25 ml). Theresulting solid was collected by filtration, washed with MTBE and driedin vacuo to give 6.31 g of a second crop of crude methyl2-ethoxy-5-nitrobenzoate. The combined portions of methyl2-ethoxy-5-nitrobenzoate were taken up in hot IPA (50 ml) and cooled toRT. The resulting solid was collected by filtration, washed with MTBEand dried in vacuo to give pure methyl 2-ethoxy-5-nitrobenzoate (5.49 g,40.5%) as a yellow solid.

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.38 (3H, t, CH₃CH₂O), 3.85 (3H, s, OMe),4.28 (2H, q, CH₃CH₂O), 7.38 (1H, d, aromatic), 8.40 (1H, dd, aromatic),8.48 (1H, d, aromatic).

Step 2

Methyl 2-ethoxy-5-nitrobenzoate (6.099 g, 26.99 mmol) and 50% wt/vaqueous hydroxylamine (40 ml) were taken up in MeOH (100 ml) and stirredat RT overnight. The resulting precipitated yellow solid was collectedby filtration, washed with IPA and dried in vacuo. The filtrate wasevaporated in vacuo and the residue was triturated with IPA (25 ml). Thesolid that didn't dissolve was filtered off, washed with a minimumquantity of IPA and dried in vacuo. The two crops of solid weresuspended in H₂O (300 ml) and the pH was lowered to 2 by carefuladdition of concentrated HCl. The solid was filtered off, washed withMTBE and dried in vacuo to give 3.9 g of crude2-ethoxy-5-nitro-N-hydroxybenzamide. The IPA mother liquors from abovewere combined with the crude 2-ethoxy-5-nitro-N-hydroxybenzamide andevaporated in vacuo. The residue was triturated with CH₂Cl₂ (25 ml) andthe solid was filtered off and dried in vacuo to give2-ethoxy-5-nitro-N-hydroxybenzamide (2.39 g, 39%) as a yellow solid.

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.39 (t, 3-H, CH₃CH₂O), 4.28 (q, 2-H,CH₃CH₂O), 7.34 (d, 1-H, aromatic), 8.38 (dd, 1-H, aromatic), 8.48 (d,1-H, aromatic), 9.32 (s, 1-H, NH), 10.79 (s, 1-H, OH).

Step 3

2-Ethoxy-5-nitro-N-hydroxybenzamide (2.39 g, 1.06 mmol) was suspended inEtOH (50 ml) and 10% Pd on carbon (wet basis) (240 mg) was added. Themixture was hydrogenated at 50 psi for 1.5 hrs. MeOH (50 ml) was addedand the mixture was filtered through celite. The volatiles were removedin vacuo and the residue was taken up in IPA (50 ml). The mixture washeated at 60° C. for 0.5 hr then stood at RT overnight. The precipitatedsolid was collected by filtration, washed with MTBE and dried in vacuoto give 1.51 g of 5-amino-2-ethoxy-N-hydroxybenzamide (73%) as anoff-white solid.

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.28 (t, 3-H, CH₃CH₂O), 3.97 (q, 2-H,CH₃CH₂O), 4.81 (s, 2-H, NH₂), 6.61 (dd, 1-H, aromatic), 6.80 (d, 1-H,aromatic), 6.87 (d, 1-H, aromatic), 9.00 (s, 1-H, NH), 10.35 (s, 1-H,OH).

EXAMPLE 5 Method of Preparing6-Amino-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one (Compound 28)

Step 1

5-Nitrosalicylic acid (25 g, 136.6 mmol) was taken up in acetone (20 ml)and trifluoroacetic acid (150 ml) and trifluoroacetic anhydride (50 ml)were added. The mixture was heated at reflux. After 1 hr more acetone(30 ml) was added, and the reaction was heated at reflux for 48 hrs. Thereaction was cooled to RT and the volatiles were removed in vacuo. Theresulting brown oil was dissolved in CH₂Cl₂ (400 ml) and washed with 1:1H₂O/saturated NaHCO₃ (400 ml). The aqueous phase was extracted withCH₂Cl₂ (2×200 ml), and the combined organic layers were dried (MgSO₄)and evaporated in vacuo. The solid residue was triturated with pentane(150 ml), collected by filtration, washed thoroughly with pentane anddried in vacuo to give 6-nitro-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one(27.84 g, 91%) as a dark yellow solid.

¹H NMR (δ, 250 MHz, CDCl₃): 1.79 (s, 6-H, CH₃), 7.13 (d, 1-H, aromatic),8.43 (dd, 1-H, aromatic), 8.87 (d, 1-H, aromatic).

Step 2

6-Nitro-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one (5 g, 22.42 mmol) wastaken up in EtOH (35 ml) and 10% Pd on C (wet basis) (2.37 g) was added.The mixture was hydrogenated at 50 psi for 1 hr. The mixture wasfiltered through celite and the volatiles were removed in vacuo. IPA (50ml) was added and the mixture was heated at 60° C. for 5 mins thenallowed to cool to RT. The resulting solid was filtered off, washed withMTBE and dried in vacuo to give 2.93 g of6-amino-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one (68%) as a yellow solid.

¹H NMR (δ, 250 MHz, CDCl₃): 1.71 (s, 6-H, CH₃), 6.80 (d, 1-H, aromatic),6.93 (dd, 1-H, aromatic), 7.26 (d, 1-H, aromatic)

EXAMPLE 6 Method of Preparing 5-Amino-2-isopropoxybenzoic Acid (Compound31)

Methyl 2-hydroxy-5-nitrobenzoate (11.82 g, 60 mmol), triphenylphosphine(17.29 g, 66 mmol) and ^(i)PrOH (3.96 g, 5 ml, 66 mmol) were taken up inTHF (150 ml) and cooled in ice. Di-isopropyl-azodicarboxylate (8.31 g,10 ml, 66 mmol) was added carefully, and the reaction was stirred at RTovernight. The volatiles were removed in vacuo and the residue treatedwith EtOAc (50 ml). The solids that didn't dissolve were removed byfiltration, and the filtrate evaporated to dryness in vacuo. The residuewas purified by column chromatography to give methyl2-isopropoxy-5-nitrobenzoate (6.92 g, 48.5%) as a yellow oil.

Methyl 2-isopropoxy-5-nitrobenzoate (6.92 g, 28.95 mmol) was taken up inTHF/H₂O (35 ml of each) and LiOH (1.39 g, 57.9 mmol) was added. Themixture was stirred at RT overnight then the pH was lowered to 1 by theaddition of concentrated HCl and the product extracted in ethyl acetate(50 ml). The organic layer was dried (MgSO₄) and evaporated to give2-isopropoxy-5-nitrobenzoic acid (6.02 g, 92.5%) as a yellow solid. ¹HNMR (δ, 250 MHz, DMSO-d₆): 1.32 (d, 6-H, CH₃), 4.88 (septet, 1-H, CH),7.37 (d, 1-H, aromatic), 8.32 (dd, 1-H, aromatic), 8.40 (d, 1-H,aromatic), 13.13 (s, 1-H, CO₂H). 2-Isopropoxy-5-nitrobenzoic acid (6.02g, 26.75 mmol) was suspended in EtOH (100 ml) and 10% Pd on C (wetbasis) (600 mg) was added. The mixture was hydrogenated at 50 psi for 2hrs. The mixture was filtered through celite and the volatiles wereremoved in vacuo. The residue was triturated with IPA, and the resultingsolid filtered off, washed with MTBE and dried in vacuo to give 4.15 gof 5-amino-2-isopropoxybenzoic acid (79.5%) as a pale yellow solid.

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.21 (d, 6-H, CH₃), 4.33 (septet, 1-H,CH), 7.68 (dd, 1-H, aromatic), 6.84 (d, 1-H, aromatic), 6.89 (d, 1H,aromatic)

EXAMPLE 7 Method of Preparing 4-Diisopropylaminosalicylic Acid (Compound38)

A mixture of 5-aminosalicylic acid (3.3 g, 21.6 mmol), 2-iodopropane(9.1 g, 53.9 mmol) and potassium carbonate (7.4 g, 54 mmol) was stirredat 50° C. in IMS (300 ml) and water (100 ml) for 4 days. Heating wasstopped and the mixture concentrated to a solid, which was washed with100 ml CH₂Cl₂. The washings were discarded and the solid heated in IMS(200 ml) for 30 minutes at 40° C. When heating was stopped, magnesiumsulphate was added and stirring continued for 25 minutes. Afterfiltration to remove the inorganic solids the solution was concentratedand purified by silica chromatography (eluent 10-20% methanol/CH₂Cl₂).This gave 0.5 μg of 4-diisopropylaminosalicylic acid.

¹H NMR (δ, 250 MHz, d6-DMSO): 1.51 (12H, brd, 5.75 Hz), 3.9-3.5 (1H,br), 4.41 (2H, br), 7.21 (1H, d, 8.8 Hz), 7.70 (1H, dd, 8.8, 2.7 Hz),8.12 (1H, brs)

EXAMPLE 8 Study on the Effects of New Compounds According to theInvention on PPARγ Activation/Expression and Regulation of CellProliferation and Apoptosis

Materials and Methods

Compounds

5-ASA was purchased at Sigma-Aldrich™ (St Quentin Fallavier, France).Rosiglitazone was acquired at Spi Bio™ (Massy, France). The newmolecules 13, 14, 17, 26, 31, 38 (FIG. 1A) were given by Giuliani SpA™(Milano, Italy) and synthesized by SAFC Pharma™ (Manchester, England).

Cell Lines

The colon carcinoma cell line HT-29 STD (ATCC HTB-38) was routinelygrown in DMEM supplemented with 10% heat-FCS, and antibiotics. Cellswere grown in monolayers, incubated at 37° C. in 5% CO₂ and 95% relativehumidity.

Transient Transfection with PPARγ and Stimulation of Cells

HT-29 STD cells were transiently transfected using the Effectene™transfection reagent (Qiagen™) according to instructions from themanufacturer. To test PPARγ activation, we performed transfection with500 ng of a minimal promoter construct containing two copies of PPREobtained from the cytochrome p450 4A (2XCYP) (1). The renilla luciferaseplasmid (0.1 g/well) was also transfected as an internal control formonitoring transfection efficiency and for normalizing the fireflyluciferase activity. Transfected cells were left for 48 hours incubationat 37° C. Stimulations were performed after incubation of cells during3-6-9-12-15-18-24 hours with the compounds 13, 14, 17, 26, 31, 38 at aconcentration of 30 mM and compared with the two PPARγ synthetic ligands5-ASA 30 mM (2) and rosiglitazone 10⁻⁵ M (2) used as positive controls.The pH of the drug solutions was adjusted to 7.4 with NaOH. Total cellextracts were prepared using the Passive Lysis Buffer (Promega™,Madison, Wis.). Luciferase activity was assayed in 20 μl of the extractusing the Promega™ Dual Luciferase assay system according to themanufacturer's protocol. Transfections were assayed in triplicate in atleast three separate experiments. The luciferase activity was expressedas fold of the activity obtained in cells treated with the differentmolecules divided by luciferase activity from non-stimulated cells.

Evaluation of PPARγ and β-Actin by Western Blot Analysis

The total proteins were obtained by cell homogenization in an extractionbuffer consisting of PBS with 2% Triton™, Phenyl Methyl SulphonylFluoride (PMSF) 100 mM and a classical protease inhibitor cocktail (2).The total proteins were then separated by polyacrylamide gelelectrophoresis and electroblotted. Polyvinylidendifluoride (PVDF)membranes were incubated overnight with rabbit polyclonal primaryantibody directed against PPARγ (dilution 1/500, TEBU, Le Perray enYveline, France). β-actin was detected using a rabbit monoclonal primaryantibody diluted at 1/10,000 (Sigma). Immunodetection with a secondaryperoxidase-conjugated antibody (1/1000, Dako™, Trappes, France) andchemiluminescence was performed according to the manufacturer's protocol(ECL™, Amersham Pharmacia Biotech™, Orsay, France). Optical densityvalues of PPARγ were given for each condition in proportion to thequantity of the internal control β-actin in the same sample (2).

Analysis of Cell Proliferation by Ki-67 Immunostaining

After 24 h of culture, HT-29 STD cells were treated during 24 h with thenew molecules 13, 14, 17, 26 and 31, at 30 mM. 5-ASA (30 mM) androsiglitazone (10⁻⁵M) were used as positive controls. The molecule 38(example 7) was not included in this experiment due to its poorsolubility. The pH of the drug solutions was adjusted to 7.4 with NaOH.Cells were fixed in PFA 4%, permeabilized in PBS containing 0.1% TritonX-100™ at 4° C. and then incubated with goat normal serum and blockingbuffer (1% BSA in PBS) to minimize non-specific adsorption of theantibody.

Cell proliferation was assessed by a nuclear Ki-67 staining using mousemonoclonal primary antibody directed against Ki-67 (dilution 1:50overnight; ZYMED™ Clinisciences™, Montrouge, France). Primary antibodywas revealed with Alexa 594 donkey anti-mouse IgG conjugated to acridinred fluorochrome (dilution 1:100, Molecular Probes™, Invitrogen™, CergyPontoise, France). Nuclei were stained with Hoescht 33342 solution(0.125 mg/mL) (Sigma-Aldrich™) and visualized under a fluorescencemicroscope (Leica™, Bensheim, Germany). Negative controls consisted ofstaining with a non-specific mouse serum instead of the specificantibody. Counts of at least 500 cells/sample were systematicallyperformed blindly in one experiment. The results were expressed as themean±SEM of the number of stained cells.

Detection of Apoptosis

After 24 h of culture, HT-29 STD cells were treated during 24 h with thenew molecules 13, 14, 17, 26 and 31, at a concentration of 30 mM. 5-ASA(30 mM) and rosiglitazone (10⁻⁵M) were used as positive controls. Themolecules 17 and 38 (examples 3, 7) were not included in this experimentdue to their poor solubility. The pH of the drug solutions was adjustedto 7.4 with NaOH. Cells undergoing apoptosis were identified byenzymatic labelling of DNA strands using a terminal transferase dUTPnick end labelling assay (TUNEL assay, Roche Diagnostics™, Meylan,France). Counts of at least 500 cells/sample were systematicallyperformed blindly in one experiment. The results were expressed as themean±SEM of the number of stained cells.

Results

It has been observed that the new molecules 17 (example 3) and 31(example 6) induce PPARγ activation. Compound 26 (example 4) alsoinduces PPARγ, but to a lesser extent. Activation of PPARγ results in acascade of reactions leading to a binding to specific DNA sequenceelements termed peroxisome proliferator response elements (PPRE) (7-9).

We investigated PPARγ transcriptional activity by transienttransfections of epithelial cells with the renilla luciferase PPREplasmids. Cells were stimulated with the different molecules during 24hours. Analysis of PPARγ activity in transfected HT-29 cells showed thatthe new molecules 17 (example 3) and 31 (example 6) at a concentrationof 30 mM increased the reporter gene activity by two-fold therebydisplaying an activity similar to 5-ASA and rosiglitazone (FIG. 1B).Molecules 13, 14 and 38 (examples 1, 2, 7) at a concentration of 30 mMexerted a rapid cytotoxic effect on epithelial cells limiting theinvestigation of PPARγ activation after 6 hours (FIG. 1B).

The new molecules 17, 26 and 31 induce PPARγ expression. The capacity ofnew molecules to induce PPARγ expression at the protein levels in theHT-29 cell line. A mean 2-fold induction of PPARγ protein levelsquantified by western blot was observed in cells treated during 24 hourswith the molecules 17, 26 and 31 (FIG. 2).

The new molecules 17 and 31 (examples 3 and 6) inhibit epithelial cellproliferation. We evaluated in HT-29 STD cell line the role of the newmolecules in the regulation of cell proliferation (FIG. 3). Cellproliferation was assessed by nuclear protein Ki-67 staining expressedin proliferating cells, the presence of Ki-67 being necessary tomaintain cell proliferation (10). Compared to untreated cells,incubation of HT-29 cells for 24 h with the molecules 17 and 31 (30 mM)resulted in a 67 to 75% inhibition of cell proliferation (FIG. 3). Thecell proliferation effects of compound 26 were not tested, since it hasa lesser effect as a PPAR-gamma activator.

Similar results were obtained with the two positive controlsrosiglitazone (10⁻⁵M) and 5-ASA (30 mM) used at their optimalconcentrations. Demonstration of the potential anti-mitogenic effect ofthe molecules 13, 14 and 26 (examples 1, 2, 4) was limited by theirrapid cytotoxic effects on epithelial cells at this concentration (datanot shown).

The new molecule 31 (example 6) induces epithelial cell apoptosisthrough PPARγ. Similarly to rosiglitazone and 5-ASA, the molecule 31displayed apoptosis in 80% of epithelial cells identified by labellingDNA strand breaks using a terminal transferase dUTP nick end labelling(TUNEL) (FIG. 4). Similarly to the previous experiment, molecules 13, 14and 26 induced a rapid cytotoxic effect at 30 mM impeding cell apoptosisanalysis.

EXAMPLE 9 Study on the Effects of New Compounds on PPARγ Activation

Materials and Methods

Compounds

5-ASA was purchased at Sigma-Aldrich™ (St Quentin Fallavier, France).The new molecules 13, 14, 17, 26, 28, 31, 38 (FIG. 1A) were synthesizedas described in examples 1-7.

Cell Lines

The colon carcinoma cell line HT-29 STD (ATCC HTB-38) was routinelygrown in DMEM supplemented with 10% heat-FCS, and antibiotics. Cellswere grown in monolayers, incubated at 37° C. in 5% CO₂ and 95% relativehumidity.

Transient Transfection with PPARγ and Stimulation of Cells

HT-29 STD cells were transiently transfected using the Effectene™transfection reagent (Qiagen™) according to instructions from themanufacturer. To test PPARγ activation, we performed transfection with500 ng of a minimal promoter construct containing two copies of PPREobtained from the cytochrome p450 4A (2XCYP) (1). The renilla luciferaseplasmid (0.1 μg/well) was also transfected as an internal control formonitoring transfection efficiency and for normalizing the fireflyluciferase activity. Transfected cells were left for 24-hours incubationat 37° C. Stimulations were performed after incubation of cells during18 hours with the compounds 13, 14, 17, 26, 28, 31 and 38 (see FIG. 6)at a concentration of 1 mM and compared with the two PPARγ syntheticligands 5-ASA 30 mM (2) used as positive controls. The pH of the drugsolutions was adjusted to 7.4 with NaOH. Total cell extracts wereprepared using the Passive Lysis Buffer (Promega™, Madison, Wis.).Luciferase activity was assayed in 20 μl of the extract using thePromega™ Dual Luciferase assay system according to the manufacturer'sprotocol. Transfections were assayed in triplicate in at least threeseparate experiments. The luciferase activity was expressed as fold ofthe activity obtained in cells treated with the different moleculesdivided by luciferase activity from non-stimulated cells.

Results

Activation of PPARγ results in a cascade of reactions leading to abinding to specific DNA sequence elements termed peroxisome proliferatorresponse elements (PPRE) (7-9).

We investigated PPARγ transcriptional activity by transienttransfections of epithelial cells with the renilla luciferase PPREplasmids. To evaluate if the new molecules have more efficacy than 5-ASAto stimulate PPAPγ activation, we tested these molecules at aconcentration of 1 mM. The effect of the new molecules at aconcentration of 1 mM was compared to 5-ASA, used as a positive controlat optimal concentration of 30 mM. Cells were stimulated with thedifferent molecules during 24 hours. Analysis of PPARγ activity intransfected HT-29 cells showed that the new molecules increased thereporter gene activity thereby displaying an activity similar orsuperior to 5-ASA (concentration of the new molecules was 30 times less,see FIG. 6).

EXAMPLE 10 Study on the Effect of the Compounds on Colon Cancer CellGrowth

The following substances 13, 14, 17, 26, 28, 31 and 38 were tested fortheir ability to modulate colon cancer cell growth. No experiment wasperformed with the compound 28 as this substance was not soluble in theculture medium.

For this purpose, three human colon carcinoma cell lines (i.e. HT-29,HT-115 and DLD-1) were used. These cell types were selected on the basisof the cyclooxigenase-2 (COX-2) expression. Indeed, HT-115 cells expressa biologically active COX-2, HT-29 cells express a non-functional COX-2isoform, and DLD-1 are COX-2-deficient cells.

HT-29 and DLD-1 cells were cultured in McCoy and RPMI1640 mediarespectively, supplemented with 10% fetal bovine serum (FBS), 1%penicillin/streptomycin (P/S) and 50 μg/ml gentamycin. HT-115 werecultured in DMEM medium supplemented with 15% FBS and 1% P/S. Cells weremaintained in a humidified incubator at 37° C., in the presence of 5%CO₂.

For cell growth assays, single-cell suspensions were plated at 2×10³cells/well (4×10³ cells/well for HT115) in 96-well culture dishes inmedium containing 0.5% FBS and allowed to adhere. The non-adherent cellswere then removed, and fresh medium containing 0.5% FBS was added intoeach well. Cells were cultured in the presence or absence of thespecified substances. Each substance was dissolved as a 25 mM stocksolution in culture medium containing 0.5% FBS, and the pH of each stocksolution was adjusted to 7.4, if necessary, with NaOH. Substances wereused at a final concentration ranging from 0.5 to 10 mM. Cellproliferation was determined by measuring the incorporation of5-bromo-2′-deoxyuridine (BrdU) into DNA using a commercially availablecell proliferation kit (Roche Diagnostics™, Monza, Italy). BrdU wasadded to the cell cultures during the last 6 hours of incubation, andthe level of BrdU-positive cells was assessed after 48 h culture byenzyme-linked immunosorbent assay (ELISA).

Optical density (OD) was determined at 450 nm using an ELISA reader.Experiments were performed in triplicate and the results are reported asthe mean±standard deviation (SD).

To assess further the effect of the compound 14 on colon epithelial cellgrowth, serum-starved DLD-1 cells were incubated in 0.2 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen™,Milan Italy) at 37° C. for 30 minutes, then extensively washed andcultured with or without the addition of the compound. After 2 daysculture, CFSE fluorescence was evaluated and the proportion of cellsundergoing divisions was determined, thus allowing calculation of bothprecursor frequency and proliferative index.

The effect of the compound 14 on colon epithelial cell death was alsoassessed. To this end, cells were cultured as indicated above for 2 daysand then the fraction of Annexin V (AV) and/or propidium iodide(PI)-positive cells was evaluated using a commercially available kit(Beckmann Coulter™, Milan, Italy). To evaluate whether the effect of thecompound 14 on colon epithelial cell death was dependent on caspaseactivity, cells were pre-incubated with z-VAD-fmk (40 μM), a pan-caspaseinhibitor, for 1 hour prior to adding the compound 14 (1.5 mM). Thefraction of AV and PI-positive cells was determined after 48 hoursculture as indicated above.

Results

The compounds differed in their ability to inhibit colon cancer cellgrowth. Results are shown in FIGS. 7 and 8. Table shows the percentageof inhibition of growth of DLD-1 cells by the specified compounds. Thesubstances 13, 14, 17, 26, and 38 exhibit a marked anti-proliferativeeffect. Among these substances, the compounds 13 and 14 significantlyreduced the cell growth, in a dose-dependent fashion, in each of thethree cell lines tested (FIGS. 7A and 7B). More than 90% of cell growthinhibition was seen when compounds were used at a final concentration of10 mM. The anti-mitogenic effect of the compounds 17, 26 and 38 wasobserved only at concentrations of 2.5 mM or higher (FIG. 8A, 8C, 7D).

The compound 31 significantly inhibited cell growth when used at highdoses (10 mM) (FIG. 7C).

Overall the above data indicate that substances 13, 14, and 38 are themost powerful suppressors of colon cancer cell growth. However,precipitates developed in cell cultures carried out in the presence ofthe substances 13 and 38. This was followed by a massive cell death,which makes it difficult to judge if the anti-proliferative effect ofthese compounds was due to either their anti-mitogenic action or toxiceffect. Based upon these findings, subsequent experiments focused on theuse of the compound 14.

First, the inhibitory effect of the compound 14 on the growth of DLD-1cells was confirmed by flow-cytometry. To track cell proliferation,cells were labeled with CFSE. In line with the BrdU assay data, thecompound 14 inhibited the proliferation of DLD-1 cells (FIG. 9).Importantly, after 48 hour treatment, the fraction of proliferatingcells decreased from 90±4% in untreated cultures to 48±7, 21±11 and11±6% in cell cultures treated with 0.5, 1 and 2.5 mM.

To examine whether the compound 14 also regulated the survival of coloncancer cells, DLD-1 cells were cultured in the presence or absence ofsuch a compound for 48-hours and then the percentage of Annexin V and/orPI-positive cells was evaluated by flow cytometry. As shown in FIG. 10A,compound 14 significantly enhanced the fraction of cell death when usedat a final concentration of 1.5 or 3 mM. To explore the involvement ofcaspases in the 14-mediated DLD-1 cell death, cells were preincubatedwith a pan-caspase inhibitor, Z-VAD, and then treated with 14. As shownin the representative experiment in FIG. 10B, treatment of cells withZ-VAD largely reduced the percentage of AV/PI-positive cells.

The percentage of DLD-1 cell inhibition by graded doses (0.5-10 mM) ofthe specified compounds is present in Table 1. Cells were cultured inthe presence or absence of the compounds, and cell growth was thenassessed by the colorimetric (BrdU) assay after 48-hours culture.

EXAMPLE 11 Molecular Modelling

Molecular modelling studies were performed using SYBYL software version6.9.1 (Tripos Associates Inc™, St Louis, Mo.) running on SiliconGraphics™ workstations. Three-dimensional model of the zwitterions formof 5-ASA was built from a standard fragments library, and its geometrywas subsequently optimized using the Tripos force field (3). As thepK_(a) of compounds is still unknown, the SPARC online calculator wasused to determine the species occurring at physiological pH (7.4)(http://ibmlc2.chem.uga.edu/sparc/index.cfm). Three-dimensional modelsof ionized compounds were built from a standard fragments library, andtheir geometry was subsequently optimized using the Tripos force field(3) including the electrostatic term calculated from Gasteiger andHuckel atomic charges. The method of Powell available in Maximin2procedure was used for energy minimization until the gradient value wassmaller than 0.001 kcal/mol.Å. The structure of the human PPARγligand-binding domain was obtained from its complexed X-Ray crystalstructure with the tesaglitazar (AZ 242) available in the RCSB ProteinData Bank (1I7I) (4,5). Flexible docking of the compounds into thereceptor active site was performed using GOLD software (6). The moststable docking models were selected according to the best scoredconformation predicted by the GoldScore (6) and X-Score scoringfunctions (7). The complexes were energy-minimized using the Powellmethod available in Maximin2 procedure with the Tripos force field and adielectric constant of 4.0 until the gradient value reached 0.01kcal/mol.Å. The anneal function was used defining the ligand a hotregion (10 Å).

Docking Studies

All new molecules fit tightly with the PPARγ-LBD interacting viahydrogen bonding with His-323, His-449, Tyr-473 and Ser-289 consideredas key determinants required for molecular recognition and PPARγactivation (11-12) (FIG. 5A, 5C, 5D). Docking for 5-ASA is shown in FIG.5B.

CONCLUSIONS

It has previously shown that anti-inflammatory effects of 5-ASA weremediated through PPARγ mainly expressed in the colon by epithelial cells(2). The rational development of the first 6 new optimized 5-ASAmolecules based on docking analysis revealed that the 2 molecules 31 and17, used at a concentration of 30 mM, activate PPARγ and induce itsexpression by intestinal epithelial cells. These 2 new molecules alsoinhibit epithelial cell proliferation and induce apoptosis, twoimportant mechanisms involved in the development of colonic cancer andattributed to PPARγ activation. Concerning the 4 other molecules (13,14, 26, 38), most of them have direct cytotoxic effects on epithelialcells at a concentration of 30 mM, impeding the analysis of PPARγactivation and regulation and evaluation of cell proliferation andapoptosis.

This first set of examples of the invention (Example 8) shows theability of two optimized molecules 31 and 17 to stimulate PPARγexpression and activation and to regulate epithelial cell proliferationand apoptosis. The cytotoxic effects on epithelial cells of compounds13, 14 and 26 at 30 mM may be related to the presence in their structureof a highly reactive hydroxamic acid group known to display a greataffinity for many various enzymes.

The second set of examples of the invention (Example 9) indicates thatalso the other test the molecules display an activity similar to, orsuperior to 5-ASA at a working concentration thirty times less than thatof 5-ASA, over a period of 24 hours.

The final set of examples of the invention (Example 10) shows that thecompounds affect the inhibition of the growth of the colon cancer celllines, HT-29, HT-115 and DLD1 to varying degrees, with compounds 13, 14and 38 showing the highest effects. In order to clarify the nature ofthe cell death, compound 14 was further investigated and was confirmed,by flow-cytometry, to inhibit the growth of DLD-1 colon cancer cellswith increasing concentration over time.

These molecules are also active on cells that do not express COX-2, andthus the molecules of the present invention may be used in cells whichdo not express COX-2 for the purposes of treating tumours and otherapplications as herein described.

OVERALL CONCLUSIONS

The synthesized highest ranking compounds, indicated from modellingstudies, all show an activity similar/superior to that of mesalazine.

REFERENCES

-   1. Dubuquoy, L., E. A. Jansson, S. Deeb, S. Rakotobe, M.    Karoui, J. F. Colombel, J. Auwerx, S. Pettersson, and P.    Desreumaux. 2003. Impaired expression of peroxisome    proliferator-activated receptor gamma in ulcerative colitis.    Gastroenterology 124:1265-1276.-   2. Rousseaux C, Lefebvre B, Dubuquoy L, Lefebvre P, Romano O, Auwerx    J, Metzger D, Wahli W, Desvergne B, Naccari G C, Chavatte P, Farce    A, Bulois P, Cortot A, Colombel J F, Desreumaux P. Intestinal    anti-inflammatory effect of 5-amino salicylic acid is dependent on    PPARγ. J Exp Med 2005; 201: 1205-15.-   3. Clark, M. C. R. D. I. V. O., N. 1989. Validation of the General    Purpose Tripos 5.2 Field. J. Comput Chem. 10:982-1012.-   4. Gampe, R. T., Jr., V. G. Montana, M. H. Lambert, A. B.    Miller, R. K. Bledsoe, M. V. Milburn, S. A. Kliewer, T. M. Willson,    and H. E. Xu. 2000. Asymmetry in the PPARgamma/RXRalpha crystal    structure reveals the molecular basis of heterodimerization among    nuclear receptors. Mol Cell 5:545-555.-   5. Jones, G., P. Willett, R. C. Glen, A. R. Leach, and R.    Taylor. 1997. Development and validation of a genetic algorithm for    flexible docking. J Mol Biol 267:727-748.-   6. Wang, R., L. Lai, and S. Wang. 2002. Further development and    validation of empirical scoring functions for structure-based    binding affinity prediction. J Comput Aided Mol Des 16:11-26.-   7. Westin, S., R. Kurokawa, R. T. Nolte, G. B. Wisely, E. M.    McInerney, D. W. Rose, M. V. Milburn, M. G. Rosenfeld, and C. K.    Glass. 1998. Interactions controlling the assembly of    nuclear-receptor heterodimers and co-activators. Nature 395:199-202.-   8. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G.    Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon,    and et al. 1995. The nuclear receptor superfamily: the second    decade. Cell 83:835-839.-   9. Misra, P., E. D. Owuor, W. Li, S. Yu, C. Qi, K. Meyer, Y. J.    Zhu, M. S. Rao, A. N. Kong, and J. K. Reddy. 2002. Phosphorylation    of transcriptional coactivator peroxisome proliferator-activated    receptor (PPAR)-binding protein (PBP). Stimulation of    transcriptional regulation by mitogen-activated protein kinase. J    Biol Chem 277:48745-48754. Epub 42002 September 48727.-   10. Gerdes, J. et al. 1986. Growth fractions in breast cancers    determined in situ with monoclonal antibody Ki-67. J Clin Pathol 39,    977-80.-   11. Nolte, R. T., G. B. Wisely, S. Westin, J. E. Cobb, M. H.    Lambert, R. Kurokawa, M. G. Rosenfeld, T. M. Willson, C. K. Glass,    and M. V. Milburn. 1998. Ligand binding and co-activator assembly of    the peroxisome proliferator-activated receptor-gamma. Nature    395:137-14-   12. Xu, H. E., M. H. Lambert, V. G. Montana, K. D. Plunket, L. B.    Moore, J. L. Collins, J. A. Oplinger, S. A. Kliewer, R. T. Gampe,    Jr., D. D. McKee, J. T. Moore, and T. M. Willson. 2001. Structural    determinants of ligand binding selectivity between the peroxisome    proliferator-activated receptors. Proc Natl Acad Sci USA    98:13919-13924. Epub 12001 November 13916.

TABLE 1 1% DLD-1 cell inhibition by graded doses (0.5-10 mM) of thespecified compounds % of growth inhibition mM 0.5 1 2.5 5 10 13 21.1 7392.7 96.3 96.4 14 22.8 36 76.5 90.5 94.1 17 0 0 33.3 89.3 90.7 26 13.619 62 86 94.5 28 NOT SOLUBLE 31 0 0 0 8.8 31.7 38 6 11.1 51.9 85.9 94.5

The invention claimed is:
 1. A compound of the general formula (I)

in which R₁ and R₂, which may be identical or different, are selectedfrom the group consisting of —H, a linear or branched alkyl group havingfrom 1 to 6 carbon atoms, or together form an aliphatic ring; R₃ isselected from, —NHOH, —OH, —OR₆ in which R₆ is a linear or branchedalkyl group having from 1 to 6 carbon atoms; R₇, R₈ are hydrogen atoms;and R₄ and R₅, together form an aliphatic ring, fused to the benzene,with 1 to 2 heteroatoms selected independently from N or O; and saltsthereof.
 2. A compound as claimed in claim 1 wherein R₁ and R₂ are eachindependently selected from the group consisting of: —CH₃, —C₂H₅,isopropyl or propyl.
 3. A compound as claimed in claim 1 wherein R₄ andR₅ form a ring according to the following formula (III)


4. A compound selected from the group consisting of:4-amino-N-hydroxy-2-methoxybenzamide,5-amino-N-hydroxy-2-methoxybenzamide,5-amino-2,3-dihydrobenzofuran-7-carboxylic acid,5-amino-2-ethoxy-N-hydroxybenzamide, 5-amino-2-isopropoxybenzoic acid,and 5-diisopropylaminosalicylic acid.
 5. A compound according to claim 1wherein R₁ and R₂, are both —CH(CH₃)₂.
 6. A compound the represented bythe following structure:

and salts thereof.
 7. A compound according to claim 1 wherein R₁ and R₂,are both —H.
 8. A compound according to claim 1 represented by thefollowing structure


9. A compound represented by the following structure


10. A compound represented by the following structure


11. A compound represented by the following structure

and salts thereof.
 12. A compound represented by the following structure


13. A pharmaceutical composition comprising one or more compounds asdefined in claim 1 as active principles in combination with one or morepharmaceutically acceptable excipients or adjuvants.
 14. A method of ortreating colon tumours expressing the PPARγ receptors and the EGFreceptors in a human or animal in need thereof, comprising administeringto the human or animal a compound according to claim
 1. 15. A method oftreating Crohn's disease or ulcerative rectocolitis in a human or animalin need thereof, comprising administering to the human or animal acompound according to claim
 1. 16. A method of treating Crohn's disease,ulcerative rectocolitis or colon cancer in a human or animal in needthereof, comprising administering to the human or animal a compoundselected from the group consisting of4-amino-N-hydroxy-2-methoxybenzamide,5-amino-N-hydroxy-2-methoxybenzamide,5-amino-2,3-dihydrobenzofuran-7-carboxylic acid,5-amino-2-ethoxy-N-hydroxybenzamide,6-amino-2,2-dimethyl-4H-benzo[1,3]dioxin-4-one, and salts thereof.
 17. Acompound represented by (I):

wherein: R₁ and R₂, are each independently a linear or branched alkylgroup having from 1 to 6 carbon atoms; R₃ is —NHOH; R₄ is a linear orbranched alkyl group having from 1 to 6 carbon atoms, phenyl, or benzyl;and R₅, R₇, R₈ are hydrogen atoms; and salts thereof.
 18. The compoundof claim 17 wherein R₄ is selected from the group consisting of —CH₃,—CH₂CH₃ and —CH(CH₃)₂.